Although, the storage of genetic information for all living systems is derived from the organizational string of just two base pairs (A:T and G:C), additional nucleic acid bases and base-pairs are chemically possible. We have shown that two additional bases encoding a third base pair can be applied to PCR along with the I four-base, two-base-pair system of nature. This project will focus on using this newly discovered six-base PCR system to advance and simplify ligonucleotide ligation systems for highly multiplexed genetic analysis. The three-base-pair PCR technology will be used in a genetic analysis system which first uses ligation to specifically couple sequence-specific primer sets and then PCR to amplify those ligated sets. The additional complexity provided by using three base pairs will allow the products of the proposed analysis system to be analyzed by room temperature hybridization. This analysis will achieve higher complexity and better discrimination than that possible with natural DNA alone. The goal is to simplify and advance genetic analysis technology with six-base PCR in order to lower cost and increase robustness and accuracy. If this can be demonstrated in Phase I of this project, additional chemistries should benefit from a six-base PCR system including aptamer production and DNA tagging strategies.